The research described in this application focuses on the cellular and molecular biology of two enzymes of mammalian organic and amino acid metabolism whose inherited deficiencies lead to clinically significant metabolic disease in man: cystathionine beta-synthase (CBS) and propionyl CoA carboxylase (PCC). The experiments proposed are designed to advance our understanding of human inborn errors from the biochemical and cellular level to the molecular level. The proposed research uses fibroblast lines with genetically and biochemically determined defects in these enzymes. It further utilizes reagents developed toward this goal such as cDNAs, genomic sequences, antibodies, and expression vectors. These studies are aimed at 1) developing methods to rapidly and accurately localize mutations and polymorphisms in RNA and DNA isolated from patient materials; 2) characterizing mutations in CBS in our bank of cell lines from patients with homocystinuria to improve our ability to diagnose this disease as well as provide a means for heterozygote detection and to extend our knowledge about coenzyme binding (pyridoxine responsiveness), subunit interaction, enzyme activation as well as regulation of the sulfur metabolism; 3) establishing the exon/intron organization of PCC genes and examining the mutations in the alpha and beta subunits of PCC to provide correlations between certain mutations and particular phenotypes such as instability of the subunit, abnormal protein translocation, and subunit interaction, and to elucidate the basis for complementation in PCC deficiency; 4) determining the coenzyme binding site in CBS and to clarify the role of biotinylation in the transport and assembly of PCC; 5) expressing CBS in E. coli to provide a rapid test for expression of the mutant genotype and for accumulation of sufficient enzyme quantities for X-ray cystallography. Specific techniques employed will include: protein purification by both conventional and affinity techniques; immunoprecipitation; preparation of RNA and genomic DNA from patient cells; Souther, Northern and Western blots; and polymerase chain reaction amplification, cloning, double- and single-stranded DNA analysis, DNA sequencing. These studies should provide new information on both the role of individual mutations in the pathogenesis of these inborn errors and on normal mechanisms of metabolism and homeostasis in man.